KNOCK-OUT OF K+ CHANNEL IN PITUITARY--PROLACTIN CONTROL AND FEMALE FERTILITY

垂体K通道的敲除--催乳素控制与女性生育力

• 批准号: 6108919
• 负责人: KAREN Ann GREGERSON
• 金额: $ 13.96万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6108919
• 关键词: Xenopus oocyte; cooperative study; dopamine; estrogens; female; female reproductive system; fertility; gene mutation; gene targeting; genetically modified animals; hormone regulation /control mechanism; immunocytochemistry; laboratory mouse; luteinizing hormone; molecular cloning; pituitary gland; potassium channel; prolactin; reproduction; secretion; voltage /patch clamp

Prolactin (PRL), an anterior pituitary hormone, has profound effects on female fertility. In fact, hypersecretion of PRL is the major neuroendocrine-related pathology associated with female infertility. Unfortunately, the underlying defects which lead to the hypersecretion of PRL remain ill-defined. To provide a rational framework for understanding these defects and treating associated pathologies, it is essential to elucidate the cellular and molecular mechanisms of PRL secretion regulation. Normally, PRL secretion is under tonic inhibition by hypothalamic dopamine (DA). Periodic, physiological surges of PRL in females require a withdrawal from this dopaminergic inhibition. Since reduced responsiveness to DA is a hallmark of hyperprolactinemic syndromes, our attention has focused on the cellular and molecular basis of DA action on the lactotrope. We have discovered and extensively characterized a DA-activated inwardly- rectifying potassium channel (K/DA) in normal lactotropes. K/DA activation by DA leads to hyperpolarization of the lactotrope membrane and cessation of calcium-dependent action potentials, the driving force for tonic PRL secretion. In vitro studies have demonstrated a critical role for this D/DA channel in the regulation of PRL release by DA. Furthermore, the functional expression of D/DA is dependent upon estrogen, which may explain a long-recognized modulatory action of estrogen on this system. In these regards, the D/DA channel lies at the top of a hierarchy of events in dopaminergic regulation of PRL secretion. As an initial step in elucidating the molecular basis for the physiological role of K/DA in lactotropes, we have cloned three different K channel gene products from female anterior pituitary tissue. Based on functional similarities between the native channel and recombinant channels expressed in Xenopus oocytes, two of the gene products are excellent candidate subunits encoding a heteromultimeric K/DA. As a logical extension of these studies, we propose to evaluate the role of this effector K channel within its physiological context-the whole animal- through transgenic technology. To this end, we will design and construct "dominant-negative" mutants, capable of inhibiting wild-type K/DA function in lactotropes. We will then create a transgenic mouse model in which expression of this mutant is directed to pituitary lactotropes using the PRL promoter. These studies represent a timely and important extension of the P.I.'s work and will ultimately provide insight into the basis of disorders in PRL secretion and their impact on female fertility.

催乳素（PRL）是一种垂体前叶激素， 女性生育能力事实上，PRL分泌过多是主要的 与女性不孕症相关的神经内分泌相关病理学。 不幸的是，潜在的缺陷，导致高分泌的 PRL仍然不明确。提供一个理性的框架来理解 这些缺陷和治疗相关的病理，这是必不可少的， 阐明PRL分泌的细胞和分子机制 调控正常情况下，PRL分泌受到以下因素的紧张性抑制： 下丘脑多巴胺（DA）。PRL的周期性生理性激增， 雌性需要从这种多巴胺能抑制中退出。以来 对DA的反应性降低是高催乳素血症的标志 综合征，我们的注意力集中在细胞和分子基础上 DA对催乳素的作用。 我们已经发现并广泛表征了DA激活的内在- 整流钾通道（K/DA）。K/DA激活 导致催乳细胞膜超极化， 钙依赖性动作电位，紧张性PRL的驱动力 分泌物体外研究表明， D/DA通道在DA调节PRL释放中的作用 D/DA的功能性表达依赖于雌激素， 解释了雌激素对该系统的长期调节作用。在 在这些方面，D/DA通道位于事件层次结构的顶部 在多巴胺能调节PRL分泌中的作用。 作为阐明该分子基础的第一步， K/DA在催乳素中的生理作用，我们克隆了三种不同的 女性垂体前叶组织中钾通道基因产物。基于 天然通道和重组通道之间的功能相似性 在非洲爪蟾卵母细胞中表达的通道中，两种基因产物是 编码异源多聚体K/DA的优秀候选亚基。 这些研究的逻辑延伸，我们建议评估的作用， 这个效应钾通道在它的生理环境中-整个动物- 通过转基因技术。为此，我们将设计和建造 “显性阴性”突变体，能够抑制野生型K/DA功能 在催乳素中。然后我们将建立一个转基因小鼠模型， 该突变体的表达是针对垂体催乳素， PRL启动子。这些研究是对《公约》的及时和重要的延伸， 私家侦探的工作，并将最终提供洞察的基础， PRL分泌紊乱及其对女性生育力的影响。